Ethanol withdrawal in the rat: involvement of noradrenergic neurons

Ethanol dependence was induced in rats by maintaining them for 3 weeks on a liquid diet containing ethanol. When ethanol was abruptly replaced with sucrose in the diet, animals showed withdrawal symptoms. Eight hours later, the accumulation in brain and heart of ³H-norepinephrine synthesized from ³H-tyrosine, and of ³H-norepinephrine metabolites was greater than in animals not undergoing withdrawal. An injection of ethanol (3 g/kg) 1 $\text{1}\text{2}$ or 5 hours after the initiation of withdrawal resulted in less accumulation of newly synthesized ³H-norepinephrine and of ³H-norepinephrine metabolites in both brain and heart. If the rate of ethanol withdrawal was slow, i.e., the ethanol in the diet was replaced gradually with sucrose over a 3-day period, less accumulation of ³H-norepinephrine and ³H-norepinephrine metabolites occured in heart and brain than as a result of abrupt withdrawal. Also, no behavioral symptoms of withdrawal were observed. These results indicate that (a) gross withdrawal symptoms and the accompanying activation of noradrenergic neurons can be blocked during withdrawal by an acute dose of ethanol, and (b) ethanol withdrawal can be modified by altering the rate of withdrawal, a finding that may prove useful in clinical situations. We conclude that the withdrawal symptoms and the activation of noradrenergic neurons during withdrawal are caused by the sudden lack of ethanol in the system.     

Norepinephrine turnover in the rat pineal gland. Acceleration by estradiol and testosterone.

The administration of 0.5 mg of testosterone propionate (TP) to orchiectomized rats or of 2 ug of estradiol to oophorectomized rats resulted in significantly less ³H-norepinephrine remaining in the pineal gland 60 and 120 min after a pulse injection of the radioactive compound. This effect was not observed in animals administered with ³H-norepinephrine 45 or 180 min after a single hormone injection. Half-lives for ³H-norepinephrine disappearance in estradiol- and TP-treated rats were 62 and 60 min respectively whereas those of female and male vehicle-injected controls were 109 and 123 min respectively. Hormone treatment did not affect pinela norepinephrine content in any of the schedules used. Norepinephrine efflux expressed as pg. mg tissue⁻¹. min⁻¹ was 33 in estradiol-treated females, 17 in vehicle-treated females, 28 in TP-treated males and 12 in vehicle-treated males. These data indicate that the norepinephrine turnover in neurons innervating the pineal gland is increased by chronic administration of female and male sex hormones.     

Control of Diapause by Acidic pH and Ammonium Accumulation in the Hemolymph of Antarctic Copepods

Life-cycles of polar herbivorous copepods are characterised by seasonal/ontogenetic vertical migrations and diapause to survive periods of food shortage during the long winter season. However, the triggers of vertical migration and diapause are still far from being understood. In this study, we test the hypothesis that acidic pH and the accumulation of ammonium (NH₄ ⁺) in the hemolymph contribute to the control of diapause in certain Antarctic copepod species. In a recent study, it was already hypothesized that the replacement of heavy ions by ammonium is necessary for diapausing copepods to achieve neutral buoyancy at overwintering depth. The current article extends the hypothesis of ammonium-aided buoyancy by highlighting recent findings of low pH values in the hemolymph of diapausing copepods with elevated ammonium concentrations. Since ammonia (NH₃) is toxic to most organisms, a low hemolymph pH is required to maintain ammonium in the less toxic ionized form (NH₄ ⁺). Recognizing that low pH values are a relevant factor reducing metabolic rate in other marine invertebrates, the low pH values found in overwintering copepods might not only be a precondition for ammonium accumulation, but in addition, it may insure metabolic depression throughout diapause.     

The effects of phencyclidine on the uptake of 3H-catecholamines by rat striatal and hypothalamic synaptosomes

In tests of the effects of the psychotomimetic agent, phencyclidine, 1-(1-phenylcyclohexl) peperidine (PCP), on the uptake of ³H-catecholamines by synaptosome-rich homogenates of rat striatum and hypothalamus, PCP was shown to be a potent, competitive inhibitor of both ³H-dopamine uptake in the striatal region and ³H-norepinephrine uptake in the hypothalamus. The implication of these findings are discussed in terms of a drug-induced imbalance of the catecholaminergic-cholinergic systems in those regions of the brain where activity has been correlated with emotional expression.     

Acid-base regulation during ammonium assimilation in Hydrodictyon africanum

Abstract The acid-base balance during ammonium (used to mean NH ₄⁺ and/or NH₃) assimilation in Hydrodictyon africanum has been measured on cells growing with about 1 mol m^?3 ammonium at an external pH of about 6.5. Measurements made included (1) ash alkalinity (corrected for intracellular ammonium) which yields net organic negative charge, (2) the accumulation of organic N in the cells and (3) the change in extracellular H⁺ (from the pH change and the buffer capacity). These measurements showed that some 0.25 excess organic negative charge (half in the cell wall, half inside the plasmalemma) accumulates per organic N synthesized, while some 1.25H⁺ accumulate in the medium per organic N synthesized. Granted a permeability (PNH₃) of some 10^?3 cm s^?1, and a finite [NH₃] in the cytoplasm of these N-assimilating cells it is likely that most of the ammonium entering these growing cells is as NH ₄⁺. This means that most of the H ⁺ appearing in the medium must have originated from inside the cell and have been subjected to active efflux at the plasmalemma: H⁺ accumulates in the medium equivalent to any NH₃ entry by requilibration from exogenous NH ₄⁺. The cell composition (net organic negative charge, organic N content) is very similar in these ammonium-grown cells to that of NO₃⁺grown cells, suggesting that there is no action of a ‘biochemical pH stat’ during longterm assimilation of NO₃⁺in H. africanum. Short-term experiments were carried out at an external pH of 7.2 in which ammonium at various concentrations were supplied to NO₃⁺-grown cells. There was in all cases a rapid influx followed by a slower uptake; at least at the lower concentrations (less than 100 μmol dm^?3) the net influx was all attributable to NH₄⁺influx via a uniporter, probably partly short-circuited by a passive NH₃ efflux due to intrinsic membrane permeability to NH₃. The net ammonium influx was in all cases associated with H⁺ accumulation in the medium. (1.3-1.7 H ⁺ per ammonium taken up); as in the growth experiments, most of the ammonium taken up was assimilated. Determinations of cytoplasmic pH showed either no effect on, or a slight decrease in, pH during ammonium assimilation; the changes that occurred were in the direction expected for actuating a ‘pH-regulating’ change in H⁺ fluxes.     

Light and electron microscopic autoradiography of substantia nigra of rat after intraventricular administration of tritium labelled norepinephrine,dopamine, serotonin and the precursors

Summary In autoradiographies of substantia nigra in rat, it has been observed that after intraventricular injections of ³H-dopamine and ³H-norepinephrine respectively the silvergrains are accumulated in nigra neurons and their dendritic branches. The incorporation was more pronounced in the case of ³H-norepinephrine than ³H-dopamine. This seems to indicate that exogenous norepinephrine may have stronger affinity to nigra neurons and their dendrites than exogenous dopamine. In addition, some ³H-dopamine and ³H-norepinephrine labelled nerve terminals were observed in axo-dendritic synapses. In contrast to these data, ³H-5HTP and ³H-5HT administration showed almost all silver grains accumulated in the neuropil when observed in light microscopic autoradiography. Electron micrographs further reveal that the incorporation of ³H-5HTP and ³H-5HT was mostly within axo-dendritic boutons with more frequent dense core vesicles. These data again strongly suggest that substantia nigra receives a large number of serotoninergic fibres forming axo-dendritic synapses which may play an important role in modulation of substantia nigra function.Dr. Parizek was on leave of absence from the Charles University, Faculty of Medicine, Hradec Králové, Czechoslovakia.     

Bulk soil pH and rhizosphere pH of peach trees in calcareous and alkaline soils as affected by the form of nitrogen fertilizers

One-year old nectarine trees [Prunus persica, Batsch var. nectarina (Ait.) Maxim.], cv Nectaross grafted on P.S.B2 peach seedlings [Prunus persica (L.) Batsch] were grown for five months in 4-litre pots filled with two alkaline soils, one of which was also calcareous. Soils were regularly subjected to fertigation with either ammonium sulphate or calcium nitrate providing a total of 550 mg N/tree. Trees were also grown in such soils receiving only deionized water, as controls. Rhizosphere pH, measured by the use of a microelectrode inserted in agar sheet containing a bromocresol purple as pH indicator and placed on selected roots, was decreased by about 2–3 units compared to the bulk soil pH in all treatments. This decrease was slightly less marked when plants were supplied with calcium nitrate rather than ammonium sulphate or control. Measurements conducted during the course of the experiment indicated that ammonium concentration was similar in the solution of soils receiving the two N fertilizers. During the experiment, soil solution nitrate-N averaged 115 mg L^–1 in soil fertilized with calcium nitrate, 68 mg L^–1 in those receiving ammonium sulphate and 1 mg L^–1 in control soils. At the end of the experiment nitrate concentrations were similar in soils receiving the two N sources and bulk soil pH was decreased by about 0.4 units by ammonium sulphate fertigation: these evidences suggest a rapid soil nitriflcation activity of added ammonium. Symptoms of interveinal chlorosis in apical leaves appeared during the course of the experiment in trees planted in the alkaline-calcareous soil when calcium nitrate was added. The slightly higher rhizosphere pH for calcium nitrate-fed plants may have contributed to this. The findings suggest that using ammonium sulphate in a liquid form (e.g. by fertigation) in high-pH soils leads to their acidification and the micronutrient availability may be improved.     

Role of nitrification and denitrification for NO metabolism in soil

Release and uptake of NO was measured in a slightly alkaline (pH 7.8) and an acidic (pH 4.7) cambisol. In the alkaline soil under aerobic conditions, NO release was stimulated by ammonium and inhibited by nitrapyrin. Nitrate accumulated simultaneously and was also inhibited by nitrapyrin.¹⁵NO was released after fertilization with¹⁵NH₄NO₃ but not with NH₄ ¹⁵NO₃. The results indicate that in aerobic alkaline cambisol NO was mainly produced during nitrification of ammonium. The results were different under anaerobic conditions and also in the acidic cambisol. There, NO release was stimulated by nitrate and not by ammonium, and was inhibited by chlorate and not by nitrapyrin indicating that NO production was exclusively due to reduction of nitrate. The results were confirmed by¹⁵NO being released mainly from NH₄ ¹⁵NO₃ rather than from¹⁵NH₄NO₃. The observed patterns of NO release were explained by the NO production processes being stimulated by either ammonium or nitrate in the two different soils, whereas the NO consumption processes being only stimulated by nitrate. NO release was larger than N₂O release, but both were small compared to changes in concentrations of soil ammonium or nitrate.(*request for offprints)     

Growth inhibition by ammonia and use of a pH-controlled feeding strategy for the effective cultivation of Mycobacterium chlorophenolicum

The inhibitory effect of ammonia on the growth of the polychlorinated xenobiotic-degrading bacterium Mycobacterium chlorophenolicum was examined. The strain is inhibited by both the ionized and nonionized forms of ammonia. At pH 6.9 50% reduction of the growth rate was observed at 6.8 g l^–1 total ammonium. For 23 experiments performed in shake-flask culture at different pH values and ammonium concentrations a growth model based on the extended Monod kinetic fits the data with a deviation of 5.3%. To overcome growth inhibition in bioreactors a pH-controlled feeding strategy was developed for effective cultivation of M. chlorophenolicum at an ammonium level below 0.3 g l^–1. The ammonium addition was controlled on-line by the stoichiometric interdependence of ammonium consumption and pH decline. With this on-line control strategy a biomass concentration as high as 26.2 g l^–1 can be achieved within less than 1 week of cultivation, compared to a biomass concentration of 15.5 g l^–1 in normal batch culture after 2 weeks of cultivation. The yield is also increased from 0.32 g to 0.43 g biomass (g glucose)^–1. The strategy developed provides an effective method for the production of biomass of M. chlorophenolicum serving as the inoculum in remediation technologies.     

3H-dopamine uptake by synaptic storage vesicles of rat whole brain and brain regions

A crude preparation of neurotransmitter storage vesicles was obtained by differential centrifugation and the ability to take up ³H-dopamine was evaluated $\text{in}$$\text{vitro}$. The uptake was highly dependent on temperature, had an absolute requirement for ATP and Mg²⁺ and was inhibited totally by reserpine. The uptake displayed saturation kinetics, with a Km of 0.26 μM at 20°. ³H-dopamine uptake was inhibited competitively by norepinephrine, with a Ki of 0.69 μM. Vesicles derived from a primarily dopaminergic region (corpus striatum) exhibited the same ratio of uptakes of ³H-dopamine/³H-norepinephrine as did those from a primarily noradrenergic region (cerebral cortex). These results indicate that viable rat brain storage vesicles can be readily prepared and used for evaluation of pharmacologic effects on ³H-dopamine uptake, and that dopaminergic and noradrenergic storage vesicles exhibit identical uptake properties.     

Ammonium ion transport—a cause of cell death

Ammonium can be transported into the cell by ion pumps in the cytoplasmic membrane. Ammonia then diffuse out through the cell membrane. A futile cycle is created that results in cytoplasmic acidification and extracellular alkalinisation. Ammonium transport can be quantified by measuring the extracellular pH changes occurring in a cell suspension (in PBS) after addition of ammonium. By using this technique, in combination with specific inhibitors of various ion pumps, it was shown that ammonium ions are transported across the cytoplasmic membrane by the Na⁺K⁺2Cl^--cotransporter in both hybridoma and myeloma cells. Further, the Na⁺/H⁺ exchanger, which regulates intracellular pH by pumping out protons, was shown to be active during ammonium exposure. The viability of hybridoma cells suspended in PBS and exposed to NH _inf4 ^sup+ for only 90 min, was reduced by 11% (50% necrosis and 50% apoptosis). A control cell suspension did not loose viability during this time. Turning off the activity of the Na⁺/H⁺ exchanger (by amiloride) during ammonium exposure decreased viability further, while inhibiting transport itself (by bumetanide) restored viability to the same level as for the control experiment with bumetanide alone. These results show that one effect of ammonia/ammonium on cell physiology is specifically related to the inward transport of ammonium ions by membrane bound ion pumps.Abbreviations q _pH specific rate of pH increase (pH units per min and 10⁶ cells per ml)     

Human erythrocyte ammonium transport is mediated by functional interaction of ammonium (RhAG) and anion (AE1) transporters

The ammonia/ammonium (NH₃/NH ₄ ⁺ ) influx into red blood cells (RBCs) is mediated by surface glycoprotein RhAG that forms a structural complex with anion exchanger 1 (AE1, band 3). Owing to the activity of this complex, RBCs exposed to the isosmotic ammonium buffer swell and finally lyse. Isoosmotic NH ₄ ⁺ -containing media alters the pH gradient in RBCs (intracellular alkalosis in response to NH₃/NH ₄ ⁺ influx) and triggers the AE1 activity resulting in redundant chloride and water influx and finally in cell swelling. Here we demonstrate that the ammonia/ammonium transport in human RBCs depends on the pH (pH optimum 7.4 ± 0.1), temperature (Q₁₀ 2.6 ± 0.3), HCO ₃ ^? concentration (EC₅₀ 4.7 ± 0.3 mM), and AE1 function. The data confirm functional interactions between AE1 and RhAG. The initial velocity of cell swelling increased almost 50-fold in the isosmotic ammonium buffer containing 25 mM HCO ₃ ^? (37°C) in comparison to the reaction in the same buffer without HCO ₃ ^? . This indicates that the reaction is facilitated mostly by the carrier proteins, not just owing to the simple diffusion of NH₃ across the erythrocyte membrane. We demonstrate that pH_i reaches its maximum value much faster than the volume increase does. These data suggest that there is no direct correlation between pH_i changes and the influx of NH₃/NH ₄ ⁺ . Taken together, our data show that the RhAG and AE1 complex activity enables erythrocytes to be ammonia/ammonium storage sites in order to maintain the physiological blood ammonia/ammonium equilibrium.     

Effect of muscarinic agonists on the release of 3H-norepinephrine and 3H-dopamine by potassium and electrical stimulation from rat brain slices

The effect of acetylcholine (ACh) on the release of ³H-norepinephrine (NE) from the cerebellum and ³H-dopamine (DA) from the striatum following the administration of potassium chloride or electrical field stimulation was studied in superfused brain slices. ACh in conc. of 10^?6 and 10^?5M significantly inhibited the release of ³H-NE from cerebellar slices and ³H-DA from striatal slices following 2 min infusion of 50mM potassium chloride. In addition ACh produced a dose-dependent inhibition of the release of ³H-DA from striatal slices following electrical stimulation. The results obtained in the present study are quite consistent with the concept that a muscarinic inhibitory mechanism may be operative on noradrenergic and dopaminergic neurons in the brain.     

Nitrate and ammonium absorption by plants growing at a sufficient or insufficient level of phosphorus in nutrient solutions

Summary Absorption of nitrate and ammonium was studied in water culture experiments with 4 to 6 weeks old plants of barley (Hordeum vulgare L.), buckwheat (Fagopyrum esculentum L. Moench) and rape (Brassica napus L.). The plants were grown in a complete nutrient solution with nitrate (5.7±0.2 mM) or nitrate (5.6±0.2 mM) + ammonium (0.04±0.02 mM). The pH of the nutrient solution was kept at 5.0 using a pH-stat. It was found that phosphorus deficiency reduced the rate of nitrate uptake by 58±3% when nitrate was the sole N source and by 83±1% when both nitrate and ammonium were present. The reduction occurred even before growth was significantly impeded by P deficiency. The inhibition of the uptake of ammonium was less,i.e. ammonium constituted 10±1% of the total N uptake in the P sufficient plants and 30±5% in the P deficient plants. The reduction of nitrate absorption greatly decreased the difference between the uptake of anions and cations. It is suggested that P deficiency reduced the assimilation of NO ₃ ^– into the proteins, which might cause a negative feedback on NO ₃ ^– influx and/or stimulate NO ₃ ^– efflux.     

The isomers of cocaine and tropacocaine: Effect on 3H-catecholamine uptake by rat brain synaptosomes

Compared to (+)-$\text{pseudo}$cocaine, (?)-cocaine was 20 times more potent in inhibiting uptake of ³H-norepinephrine (³HNE) by cortical synaptosomes and 66 times more potent with respect to ³H-dopamine (³HDA) uptake by striatal synaptosomes. Although the tropacocaine isomers were equipotent as inhibitors of ³HNE uptake in the cortex, tropacocaine was 3.9 times more potent as an inhibitor of ³HDa uptake in the striatum than $\text{pseudo}$tropococaine. A major known cocaine metabolite, benzoylecgonine failed to inhibit the accumulation of ³HNE and ³HDA by synaptosomes from the cortex and striatum, respectively. The implications of these findings in relation to the motor stimulation seen with (?)-cocaine, (+)-$\text{pseudo}$cocaine and benzoylecgonine in rats are discussed.     

Inhibition by β-carbolines of monoamine uptake into a synaptosomal preparation: Structure-activity relationships

The potency of a series of β-carboline compounds to inhibit ³H-serotonin (³H-5-HT) uptake into a synaptosomal suspension from mouse brain was studied. The $\text{in vitro}$ structure-activity study showed the tetrahydro-β-carbolines to be the most potent inhibitors compared to unsaturated β-carbolines. $\text{In vitro}$ inhibition of ³H-norepinephrine (³H-NE) and ³H-dopamine (³H-DA) uptake was determined for some tetrahydro-β-carbolines, and the degree of inhibition of uptake of these amines was less than that for ³H-5-HT (EC50s being 5–13 times those for ³H-5-HT). The tetrahydro-β-carbolines were also found to effectively inhibit ³H-5-HT but not ³H-NE or ³H-DA uptake when they were administered intra-peritoneally. These results suggest that the behavioral effects of the tetrahydro-β-carbolines which have been reported previously may be due to a relatively selective involvement of the serotonergic neurotransmitter system.     

Growth of embryogenic sweet orange callus on media varying in the ratio of nitrate to ammonium nitrogen

Embryogenic callus from Citrus sinensis (L.) Osbeck cv. Hamlin was cultured for 28 days on 20 media arranged in a 5×2×2 factorial varying in the ratio of nitrate to ammonium nitrogen, total inorganic nitrogen, and benzyladenine. Fresh weight increase of callus and final medium pH were significantly affected by total inorganic nitrogen and the ratio of nitrate to ammonium. The nitrate to ammonium ratio accounted for 55% of the variation in the fresh weight increase of the callus and 93% of the variation in the final medium pH. Varying the NO₃ ^-:NH₄ ^- ratio provided adequate pH control.Abbreviation BA benzyladenine     

Optimal pH control of batch processes for production of curdlan by Agrobacterium species

We sought an optimal pH profile to maximize curdlan production in a batch fermentation of Agrobacterium species. The optimal pH profile was calculated using a gradient iteration algorithm based on the minimum principle of Pontryagin. The model equations describing cell growth and curdlan production were developed as functions of pH, sucrose concentration, and ammonium concentration, since the specific rates of cell growth and curdlan production were highly influenced by those parameters. The pH profile provided the strategy to shift the culture pH from the optimal growth condition (pH 7.0) to the optimal production one (pH 5.5) at the time of ammonium exhaustion. By applying the optimal pH profile in the batch process, we obtained significant improvement in curdlan production (64 g L⁻¹) compared to that of constant pH operation (36 g L⁻¹). Received 24 November 1998/ Accepted in revised form 17 June 1999     

The oxygen transfer rate as key parameter for the characterization of<Emphasis Type="Italic"> Hansenula polymorpha</Emphasis> screening cultures

Screening cultures are usually non-monitored and non-controlled due to a lack of appropriate measuring techniques. A new device for online measurement of oxygen transfer rate (OTR) in shaking-flask cultures was used for monitoring the screening of Hansenula polymorpha. A shaking frequency of 300 rpm and a filling volume of 20 ml in 250-ml flasks ensured a sufficient oxygen transfer capacity of 0.032 mol (l h)^–1 and thus a respiration not limited by oxygen. Medium buffered with 0.01 mol phosphate l^–1 (pH 6.0) resulted in pH-inhibited respiration, whereas buffering with 0.12 mol phosphate l^–1 (pH 4.1) resulted in respiration that was not inhibited by pH. The ammonium demand was balanced by establishing fixed relations between oxygen, ammonium, and glycerol consumption with 0.245±0.015 mol ammonium per mol glycerol. Plate precultures with complex glucose medium reduced the specific growth rate coefficient to 0.18 h^–1 in subsequent cultures with minimal glycerol medium. The specific growth rate coefficient increased to 0.26 h^–1 when exponentially growing precultures with minimal glycerol medium were used for inoculation. Changes in biomass, glycerol, ammonium, and pH over time were simulated on the basis of oxygen consumption.     

Purification and Characterization of Extracellular Pectinolytic Enzymes Produced by Sclerotinia sclerotiorum

An exopolygalacturonase (exoPG) and an exopolymethylgalacturonase (exoPMG) produced by Sclerotinia sclerotiorum have been purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The exoPG and the exoPMG were purified 66- and 50-fold, respectively, by using a series of separation procedures that included ammonium sulfate precipitation and gel chromatography. Molecular masses of the native proteins were 68 kDa for exoPG and 140 kDa for exoPMG. The pH optima of the enzymes were about pH 5, and their optimum temperature was 45°C. Activities of both enzymes were inhibited by Hg²⁺, Zn²⁺, Cu²⁺, and p-chloromercuribenzoate. ExoPMG activity, in contrast to exoPG activity, was stimulated by Mn²⁺ and Co²⁺. ExoPMG hydrolyzed only citrus pectin, while exoPG degraded sodium polygalacturonate and, to a lesser extent, citrus pectin. The exo mode of action of the enzymes was revealed by thin-layer chromatography of substrate hydrolysates. Antibodies raised against each purified protein exhibited no cross-reaction, thus confirming the biochemical specificities of the enzymes.